DNA encoding a protein which enables selective removal of immune complexes

ABSTRACT

Disclosed is a method and a family of materials useful for removing immune complexes from blood preferentially to soluble antibodies. The material comprises analogs of proteins which bind to the Fc region of immunoglobulin. The analogs are produced by truncating or otherwise altering the amino acid sequence of the binding protein to reduce their affinity for Fc. An array of such analogs disposed about the surface of an insoluble matrix has the ability to form multiple points of attachment to the multiple Fc&#39;s in a complex so as to bind complex strongly, whereas only weak associations are developed between the Fc region of soluble IgG and individual analogs. The preferred analogs are truncated proteins homologous to a portion of the domains of Protein A or Protein G which bind with Fc. Complex may be removed from whole blood or serum using the material and conventional plasmapheresis techniques.

This is a divisional of copending application(s) Ser. No. 07/601,029field on Oct. 23, 1990, now U.S. Pat. No. 5,084,398, which isfile-wrapper continuation of U.S. Ser. No. 07/127,224 filed Nov. 20,1989 now abandoned.

BACKGROUDN OF THE INVENTION

This invention relates to novel compositions of matter useful in theselective removal of immune complexes from serum. More specifically,this invention relates to an immunosorbent material comprising pluralpolypeptide domains designed to bind immune complex with greateraffinity than free, circulating immunoglobulin.

Immune complexes have been implicated in the pathology of a number ofhuman disease states. Indeed, the serum of many individuals withautoimmune dlsease, neoplastic disease, acquired immune deficiencysyndrome, and some infectious diseases can be demonstrated to containhigh levels of circulating immune complexes. Such complexes have beenhypothesized to mediate a variety of immunologic effector functions.Removal of the complexes from circulating blood is expected to havetherapeutic benefit.

U.S. Pat. No. 4,614,513 describes a method and apparatus for removing"immunoreactive substances" from blood comprising at least "componentsof Protein A". Protein A is a cell wall component of most strains ofStaphylococcus aureus which has the capacity to bind specifically to theFc region of a number of immunoglobulin species. The native protein ispartially buried in the cell wall via its hydrophobic N-terminal segmentwhich consists of about 150 amino acid residues. The remainder of themolecule consists of five highly homologous domains designated E, D, A,B, and C, which are consecutively arranged along the polypeptide chain,each having a molecular weight of approximately 7000 daltons. Eachdomain has the capacity to independently bind one Fc region of animmunoglobulin with apparently equal affinity. This binding interactionhas an association or binding constant (K_(a)) of approximately 5×10⁷M⁻¹, which varies slightly with the pH of the buffer and with thespecies, class, and subclass of the immunoglobulin. However, Protein Ais able to bind only two immunoglobulin molecules at one time,presumably due to steric constraints.

The binding of Protein A to the Fc region of an immunoglobulin has nosignificant effect on the affinity of the immunoglobulin for itsantigen. Protein A from native and recombinant sources accordingly isuseful as an immunosorbent for a variety of diagnostic and basicresearch applications. See European Patent No. 83306500.6 andInternational Patent Application Nos. PCT/SE/83/00297 andPCT/SE83/00298. These applications disclose "Protein A-like" moleculeswith substantially the same "Protein A-like binding" or increased"IgG-binding activity".

Unrecognized in the disclosure of U.S. Pat. No. 4,614,513 is theconstraint on the method there disclosed that Protein A binds both freeand complexed immunoglobulin. Thus, Protein A cannot be used practicallyas a therapeutic reagent to selectively remove immune complexes in thepresence of uncomplexed, soluble immunoglobulin.

It is an object of this invention to provide a novel immunosorbentmaterial which is useful, for example, in the selective removal ofimmune complexes from blood or serum. Another object is to provide a DNAsequence encoding this immunosorbent polypeptide, and to provide amethod for the removal of immune complexes from serum in the presence offree, circulating immunoglobulins.

These and other objects and features of the invention will be apparentfrom the following description, drawing, and claims.

SUMMARY OF THE INVENTION

A strategy has now been devised which allows for the selective removalof immune complexes, i.e., aggregates of immunoglobulin molecules, fromserum. In a preferred aspect, the strategy utilizes the natural abilityof staphylol Protein A and its individual binding domains to bind to theFc portion of an immunoglobulin without affecting the affinity of thatimmunoglobulin for its antigen. It also takes advantage of knownrecombinant DNA manipulative techniques to alter the structure of thenative Protein A binding domain such that it has the capacity to bind tocross-linked, complexed, or aggregated immunoglobulins (complex) withgreater affinity than it binds to free, soluble immunoglobulins(sol-Ab).

In accordance with the invention, it has been discovered that materialcomprising plural copies of an analog of a binding domain of Protein Ahaving reduced affinity for sol-Ab relative to Protein A, i.e., havinK_(a) 's less than 10⁷ M-⁻¹ and preferably less than 10⁵ M-⁻¹,demonstrate a selective preference for immune complexes. The material ischaracterized by multiple interactions between plural binding domainsand plural Fc's on different immunoglobulins within the immune complex.Multiple points of attachment to an immune complex form when a singlemolecule contains plural copies of a binding domain spaced sufficientlyapart, i.e., at least about 52 angstroms, so that multiple point bindingis permitted. Alternatively, monomeric binding sites are immobilized ona solid support at a threshold concentration such that binding sites arespaced apart on the surface of the matrix permitting multiple pointattachment with complex. The K_(a) characteristic of the binding betweena single binding domain and a single Fc is quite low, but the presenceof multiple interactions characteristic of the material of the inventionresults in a molecular association having significant stability, with aneffective binding constant approaching the product of the individualconstants of the individual binding domains.

Support for the hypothesis on which the invention is based can be foundin the literature. For example, J. J. Langone and others (Langone, Adv.Immunol., 32:157-252, 1982 and Dobre et al. J. Immunol. Meth.,66:171-178, 1984) have demonstrated that soluble IgG from species whichbind weakly to Protein A (e.g. mouse and goat), interact with higheraffinity when the immunoglobulins are aggregated to form complex.Similarly, it is known that the first component of the complementcascade, Clq, has a low affinity for the Fc portion of IgG, and that itis not bound significantly to soluble IgG molecules. Because Clqmolecules are composed of six binding sites, all having identicalspecificity for Fc, it is thought that the binding of a single Clq andthree or more Fc's (associated in an immune complex) is responsible forthe formation of an extremely stable structure (Hughes-Jones, Immunol.,32:191-198, 1977; Hughes-Jones and Gardner, Mol. Immunol., 16:697-701,1979).

Multiple, reduced-binding analogs may be covalently attached to asupport matrix at a density which is more conducive to binding complexthan it is to binding sol-Ab. This attachment can be done using anappropriate amino acid capable of reacting with a heterobifunctionalcross-linker covalently attached to the matrix, which itself has noimmunoglobulin-binding capabilities.

In accordance with the invention, complexed immunoglobulins can beselectively removed from plasma in the presence of uncomplexedimmunoglobulin by separating the blood into its plasma and cellularcomponents, exposing the plasma to the immunosorbent material, andrecombining the treated plasma with the cellular component.

These and other features of the invention will be apparent from thefollowing description, figures, and claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A shows a DNA sequence and the amino acid sequence (in both singleletter and three letter code) of the FB binding domain of native ProteinA. Underlined residues indicate the alpha helical regions; starredresidues identify those which come closest to residues in the Fc regionof human IgG during binding;

FIG. 1B discloses the amino acid sequences (in single letter code) ofnative FB₅₈) and FB analogs of the invention having reduced bindingaffinities for soluble IgG, including truncated analogs FB_(TF), FB₃₆,FB₂₉, FB₄₀, and FB₄₇.

FIG. 2 shows a recombinant DNA of the invention encoding a typicalreduced-binding analog of a Protein A binding domain (FB₂₉) useful inthe invention and its amino acid sequence, including a methionineresidue (start) at the N-terminus and a Pro, Pro, Cys, Ala, Ala spacersequence used to couple the binding site to an immobilized matrix; and

FIG. 3 are graphs showing the relative binding affinities of native FBand two repesentative FB analogs for complex and sol-Ab.

DESCRIPTION

The intermolecular forces involved in the interaction of the bindingregions of Protein A and the Fc portion of IgG were studied from x-raycrystallographic data and computer generated binding energies. A complexconsisting of a Protein A binding fragment (FB) and the Fc fragment ofhuman IgG was crystallized, and the three dimensional structure wasdetermined (Deisenhofer et al., 1978, Hoppe-Seyler's Z. Physiol Chem.359:975-985; Deisenhofer, 1981, Biochemistry, 20:2361-2378). Inaddition, computer generated estimates of binding energies for theinteraction of FB and Fc were made.

These data and information permitted the construction of a working modelor rough atlas of contact points showing for each residue in FB and Fc,the atoms in the complementary molecules which are within a distance of4 angstroms. Examination of the positions of polar and charged residuesat the contact interface by computer graphics revealed that a number ofpositive and negative charges become buried at the interface of the twomolecules (i.e., in a medium of small dielectric constant), and it washypothesized that their electrostatic interaction might contributesignificantly to the binding energy. Further analysis indicated noobvious pairs of opposing charges on the complementary surfaces of themolecules, suggesting that the binding mechanism did not involve simpleneutralization of opposing charges.

The amino acid sequence of the FB region of native Protein A is shown inFIG. lA. The molecule contains two alpha helical regions which areunderlined. Crystallographic studies have revealed that these helicesare intimately associated with the Fc region of IgG during binding. Thestarred amino acids indicated in FIG. lA are those which have beendetermined to be involved in the binding most directly, i.e., to liewithin about 4 angstroms from a residue of Fc in the Fc-FB complex.

From the foregoing information, it was predicted what changes in theamino acid sequence of the FB domain of the Protein A molecule likelywould reduce its affinity for IgG, and analogs of the FB region weredesigned to test the predictions. The amino acid sequences of several ofthe analogs are presented in FIG. lB. These analogs were shown to haveaffinities for soluble IgG with K_(a) 's of between 1×10³ M⁻¹ and 5×10⁶M⁻¹.

The approach used in designing such analogs can be understood better bya review of FIG. 1B. The protein labeled FB₅₈ is the native, or wildtype, FB sequence. FB_(TF) may be produced by cleaving the native formof the protein fragment with trypsin, which cleaves at the Lys⁷ -Glu⁸bond. FB₅₈ has a K_(a) for Fc of about 5×10⁷ M⁻¹ ; FB_(TF) has a K_(a)for FC of about 5×10⁶ M⁻¹. The remaining analogs are made by expressionof an appropriate DNA. All comprise truncated analogs of the nativefragment. The approximate affinities of these analogs for Fc in litersper mole are, respectively FB₃₆, K_(a) =2×10³ ; FB₂₉, 4×10² <K_(a)<2×10³ ; FB₄₀, 6×10³ <K_(a) <2×10⁴ ; FB₄₇, K_(a) =2×10³. Oligomers ofthe various analogs comprise moieties having high affinity for complex,e.g., greater than 10⁷ M⁻¹ and preferably greater than 10⁸ M⁻¹, and lowaffinity for Fc, e.g., less than about 10⁷ M⁻¹, and preferably less than10⁵ M⁻¹. Multiple point attachment requires a spacing between bindingdomains on the oligomer of at least 52 angstroms (center to center).Multiple point interaction with comp lex will also occur if such analogsare immobilized on a surface at a sufficient density such that two ormore Fc's in a complex can react simultaneously.

As will be appreciated by those skilled in the art, the Fc affinity ofthe FB fragment does not depend on precise duplication of the amino acidsequence and encoding DNA sequence set forth in FIG. 1. Other DNAs whichencode the same amino acid sequence may be used. Fragments of the FB ofFIG. 1 comprising less than the full amino acid sequence retain somebinding activity. It is also contemplated that amino acids in thesequence may be replaced while retaining significant binding activity.Other modified amino acid sequences, including analogs of the A, C, D,or E binding domains of Protein A, and related sequences fromfunctionally similar bacterial proteins such as fragments of protein Gfrom Streptococcus species are within the scope of the presentinvention. Such species having a binding affinity of at least 5×10² M⁻¹have utility.

There is but a single binding site recognized by Protein A on each ofthe heavy chains of IgG constituting the Fc region. Complex, on theother hand, presents an array of binding sites in close proximity. Thisstructural difference is exploited to increase selectivity of thecomplex binding moiety by designing repeats or oligomers of analogs ofthe binding sites of Protein A or other structures comprising adjacentbinding domains, each of which individually have a relatively lowaffinity for Fc. Such an oligomer has a low affinity for species havinga single binding site, such as free IgG, but a higher affinity forspecies having multiple binding sites, such as complex. The affinityconstant of such oligomers is the product of the individual affinitiesK_(o), of the repeating units if binding can occur independently at eachsite, where K_(o) is the intrinsic binding constant defined using molalconcentrations of reactants. Thus, an analog having an affinity for Fcof, for example, 1×10³ M⁻¹, can be used to produce, for example, atrimer, having an affinity for the Fc of a free immunoglobulin moleculeof the same 1×10³ M⁻¹, but an affinity for complex of approximately1×10⁹ M⁻¹. If the formation of one analog-Fc binding pair interfereswith the formation of others, then the observed K_(a) will be less than(K_(a))³ but still higher than K_(a).

It should be noted that Protein A itself may be regarded as a lineararray of binding sites. However, presumably because of steric hindrance,only two sites on a single Protein A molecule can bind at the same timeunder the best of conditions, and Protein A does not bind selectively tocomplex to any significant extent.

Genes encoding these Protein A binding site analogs may be designedbased on the amino acid sequences of the analogs shown in FIG. lB, or onother known sequences of Protein A, Protein G, or other binding domains,and prepared using known recombinant DNA techniques by assembly ofsynthetic oligonucleotides or other methods known per se. The DNA andamino acid sequence of a typical FB analog is presented in FIG. 2. Itcorresponds to the structure FB₂₉ set forth in FIG. lB, and comprises astart site (MET), a Pro, Pro, Cys, Ala, Ala sequence used, through theCys residue, to bind to an immobilization matrix, and then 29 residuesof native FB, spanning the two helical regions, and including seven ofthe nine residues thought to be most important to binding.

Generally, analogs such as those shown in FIG. lB may be expressed asfusion proteins containing a leader peptide for increased expression inE. coli, followed, as exemplified by FIG. 2, by a methionine residuewhich serves as a cyanogen bromide cleavage site for release of theleader peptide, and a cysteine residue located at or close to theanalog's amino terminus to facilitate directed immobilization of thereduced-binding analog to a solid support. Of course, many otherproduction techniques would be apparent to the skilled molecularbiologist. Such constructs are designed for use in the production of animmunosorbent material comprising a matrix of inert, relativelY highsurface area particles such as Sepharose beads (cross-linked dextran) orother biologically compatible material.

Provided the analog is bound to the matrix at least at a minimalthreshold concentration, the surface density of the binding sites permitmultiple attachments to immune complexes Stated differently, unless theanalogs are disposed on a matrix at such a low density that theirspacing exceeds the distance between binding sites on comPlex, this typeof material will bind complex preferentially to free immunoglobulin.This is in contrast to the native intact Protein A molecule, which atbest permits macromolecular aggregate formation having the empiricalformula [(IgG)₄ (Protein A)₂ ]. The concentration of analog which worksbest should be determined empirically, and will depend on such factorsas the surface area of the matrix material, mode of coupling, thespecific nature of the analog used, and the size of the immune complex.

Alternatively, the reduced binding analogs may be expressed as a ProteinA-like molecule (oligomer) comprising multiple reduced binding domainsspaced far enough apart by incorporated amino acid spacer sequences topermit the formation of multiple binding pairs. In this regard, theminimal distance between the centers of two active binding sites shouldbe about 52 angstroms. These may then be immobilized to produce animmunosorbent material with the desired selectivity.

Functionally, an "immune complex" can be described as a molecularaggregate containing multiple Fc's of IgG; the aggregate or complexesmay be the result of antigen-antibody bridging, heat aggregation, orchemical cross-linking. Because of the potential instability ofantigen-antibody aggregates and heat aggregates, covalently cross-linkedaggregates of human IgG were used as a model for natural complex in thereduction to practice of the present invention. Human IgG iscross-linked with carbodiimide, and aggregates of different sizes arerecovered by gel filtration chromatography. The aggregates used in theexperiments reported here have a molecular weight of approximately600,000 daltons, equivalent to a tetramer of IgG. Prior to use, theaggregates were diluted in human serum having a low endogenous level ofcirculating immune complexes.

The invention will be understood further from the following non-limitingexamples, which are intended to be illustrative and not restrictive.

Preparation of Transformants

The molecular biologY and microbiology involved in the construction ofthe gene encoding the FB₂₉ analog of Protein A is provided as an exampleof the molecular biology and microbiology involved in a geneconstruction.

The gene coding for the FB₂₉ analog was constructed by truncation and/orsubstitution of the FB₅₈ parent gene at both the N-terminus andC-terminus. The FB₅₈ gene had previously been synthesized by ligation ofoligonucleotides. A plasmid preparation containing the DNA sequencecoding for the FB₅₈ (FIG. 1) was digested with EcoRI and MluIrestriction enzymes in preparation for the alteration of the gene at itsN-terminus. The double cut plasmid preparation was separated from singlecut and uncut molecules by polyacrylamide gel electrophoresis. Followingelectroelution, ethanol precipitation and resuspension, the EcoRI/MluIdigested plasmid was ligated with oligonucleotides synthesized with thedesired DNA sequence designed to code for the desired Protein sequence.Each end of the sequence was designed to be complementary to the EcoRIand MluI sites generated within the plasmid by these restrictionenzymes. The ligation mixture was transformed into competent E. colicells by standard microbiological procedures. Resulting colonies werescreened for the presence of the altered N-terminus by restrictionanalysis. Verification of the desired sequence was accomplished by DNAsequencing (Sanger dideoxy method).

Similarly, the C-terminus of this altered gene was truncated bydigesting the plasmid with HindIII and PstI. Oligonucleotides of thedesired sequence (designed with compatible ends for ligation into theHindIII and PstI sites) were ligated into the double-cut plasmid.Following ligation, transformation, screening, and sequencing, the finalgene sequence (FIG. 2) was prepared for expression by inserting apromoter and appropriate leader peptide to the N-terminus of the gene.

Preparation of Inoculum for Bacterial Fermentation

A frozen stock of E. coli containing the desired plasmid is inoculatedinto 59 ml of Luria broth containing 10 g/l tryptone, 10 g/l yeastextract, 5 g/l NaCl, and 1 ml/l tetracycline stock (10 mg/ml in 95%ethanol) in a 1 liter baffled shaker flask. The culture is incubated ona rotating platform at 200 rpm for 17 hr at 37° C. The fermenter isinoculated with the entire 200 ml.

Fermentation of E. coli

The above stationary phase culture is inoculated into 10 liters ofmedium consisting of 11 g/l Na₂ HPO₄, 15 g/l D-glucose, 5 g/l acidhydrolysate, 3 g/l KH₂ PO₄, 1 g/l NH₄ Cl, 0.5 g/l NaCl, 5 ml/l tracemineral mix (13.3 ml concentrated HCl, 5.4 g/l FeCl₃.6H₂ O, 1.44 g/lZnSO₄.7H₂ O, 1.0 g/l MnCl₂.4H₂ O, 0.25 g/l CuSO₄.5H₂ O, and 0.062 g/l H₃BO₃), 0.5 ml/l 1M MgSO₄.7H₂ O, 1.4 ml/l 1M CaCl₂.2H₂ O, 0.2 ml/l 1M Na₂MoO₄.2H₂ O, 0.5 ml/l tryptophan (10 mg/ml in 100 mM acetic acid), 1.0ml/l tetracycline (10 mg/ml), 2.5 ml/l niacin (10 mg/ml), 2.5 ml/lbiotin (0.5 mg/ml in 95% ethanol) and 0.2 ml/l antifoam, pH 7.0 in a 14liter fermenter. The bacterial culture is agitated at 700 rpm andincubated at 35° C. The pH of the culture medium is maintained in therange of 6.85 to 7.15 by the addition of NH₄ OH. The culture is spargedwith filtered air at a flow rate of 10 liters per minute.

The culture is induced for expression of the protein by addition of3B-indoleacrylic acid (IAA) to a final concentration of 20 mg/ml culturewhen the absorbance at 600 nm is approximately 4. The culture is induceda second time at 20-22 hours post-inoculation to a final concentrationof 20 mg/ml IAA. At ten hours post-inoculation, the cells are fed with asolution of 500 g/l D-glucose, 2 g/l casamino acids, 50 ml/l tracemineral mix (see above), 5 ml/l 1M MgSO₄.7H₂ O, 14 ml/l 1M CaCl₂.2H₂ O,2 ml/l 1M NaMoO₄.2H₂ O, 20 ml/l biotin stock solution (see above) and 20ml/l niacin stock solution (see above), at a flow rate of 125 ml/hr. Thefeed is on for 0.5 hours and off for 2.5 hours consecutively until thefermentation is terminated at 30 hours post-inoculation.

At the end of the fermentation, the culture is decanted into a 20 litercarboy and concentrated to 1 liter with an Amicon hollow fiberultrafiltration unit. After diafiltration with 5.0 1 of deionized water,the cells are pelleted by centrifugation at 11,300×g for 10 min. Afterdecanting the supernatant, the cell pellet is transferred to appropriatecontainers and stored at -70° C.

Preparation of Inclusion Bodies

One hundred grams of frozen cell paste is resuspended in 1 liter ofdeionized water. The cells are lysed in a homogenizer operating at 5000psi. The partially lysed cells are stored of ice for 15 minutes and arepassed through the homogenizer a second time under identical conditions.Inclusion bodies and cell debris are pelleted by centrifugation at3500×g for 30 min at 4° C.

Purification of Analogs

The fusion protein is solubilized from the inclusion bodies in bufferconsisting of 40 mM Tris-HCl, 1 mM EDTA and 8M urea, pH 8.0. A volume of25 ml of buffer for each gram of inclusion bodies is added;solubilization is facilitated by stirring and homogenization. Once thefusion protein is in solution, the urea is removed by dialysis against 1mM EDTA pH 8.0 in dH₂ O overnight at 4° C. The dialyzed material isadjusted to 0.1M HCl by the addition of concentrated acid, and cyanogenbromide (0.25 g/g cell paste) is added to cleave the Protein A analogfrom the leader peptide. The reaction is incubated, with stirring, for4-6 hr in the dark at room temperature. Unreacted cyanogen bromide andvolatile by-products are removed by lyophilization. The residue isresuspended in deionized water, and the pH of the solution is adjustedto 8.0 by the addition of 1 N NaOH. After stirring at room temperaturefor 2 hr while maintaining the pH, the digest is dialyzed overnightagainst 20 mM Tris-HCl, lmM EDTA, pH 8.0. The digest is reduced by theaddition of 1 mM dithiothreitol (DTT) prior to ion exchangechromatography.

The digest is then chromatographed on an anion exchange columnconsisting of Whatman DE-52 cellulose equilibrated in 20 mM Tris-NaCl, 1mM EDTA, and 1 mM DTT, pH 8.0 (column buffer). The sample is loaded incolumn buffer. Protein eluting from the column is monitored at 280 nmand collected in 20 ml fractions. Bound proteins are eluted using agradient of 0-500 mM NaCl in column buffer. Individual fractions areevaluated for the presence of the desired analog by analytical C-18reverse phase high performance liquid chromatography (HPLC), sodiumdodecyl sulfate-polyacrylamide gel elecrophoresis (SDS-PAGE) and/orradioimmunoassay (RIA) using chicken anti-Protein A antibody (describedbelow). The appropriate fractions are pooled, concentrated, and dialyzedagainst 1 mM EDTA in distilled water. The sample is again reduced with 1mM DTT and loaded onto a preparative C18 column equilibrated in 25%acetonitrile in dH₂ O adjusted to pH 2.0 with trifluoroacetic acid.Bound material is eluted from the column using a 25-45% gradient ofacetonitrile. Identification of the analog is confirmed by co-elutionfrom an analytical C18 column with an aliquot of a previously analyzedlot of recombinant analog and/or amino acid analysis and sequencing. Thepurity of the analog is assessed by analytical reverse phasechromatography Fractions having the desired purity are pooled andlyophilized.

RIA for Protein A analogs

A "sandwich" radioimmunoassay (RIA) employing chicken anti-FB has beenused to identify and quantitate reduced binding analogs in samplesgenerated during purification. Briefly, chicken anti-FB, diluted to aconcentration of 2.5 μg/ml in borate buffered saline, pH 8.0 (BSB), isadhered to the wells of polyvinyl chloride microtiter plates byincubation at 37° C. for 1 hr in a humid atmosphere. The unbound proteinis removed, and the remaining protein binding sites are blocked byincubation of the plates with 1% nonfat skim milk in BSB for 1 hr at 37°C. Varying dilutions of Protein A analogs of known concentration orunknown samples are added to the wells for 4-18 hr at room temperature.Upon completion of the binding period, unbound proteins are removed bywashing individual wells with BSB. ¹²⁵ 1-labeled chicken anti-FB, havinga concentration of 2.5 μg/ml and a specific activity of 2500 cpm/ng, isadded to each well to detect bound analogs. The plates are incubatedovernight at room temperature, washed, and the radioactivity determined.A standard curve is drawn by plotting cpm bound per well versusconcentration of analog. The concentration of analog within an unknownsample is determined from the linear portion of the curve. Thesensitivity of this assay is 5-100 ng/ml for the native FB molecule and5-100 μg/ml for the reduced binding analogs.

Competitive RIA to Assess Analog Binding to IgG

Human IgG, diluted to a concentration of 20 μg/ml in BSB, is adhered tothe wells of polyvinyl chloride microtiter plates. The remaining proteinbinding sites are blocked by the addition of 1% skim milk in BSB. Excessprotein solution is discarded, and dilutions of FB or its analogs,having known protein concentrations, are added to the wells. Afterincubation for 30 min, a constant quantity of ¹²⁵ I-labeled FB dilutedto 0.05 μg/ml is added to each well. The plates are incubated overnightat room temperature in a humidified atmosphere. Plates are washed toremove unbound radioactivity, air dried, and the individual wells arecut and counted in a gamma scintillation spectrometer. Values for %Inhibition are calculated at each analog concentration using thefollowing formula: ##EQU1## where cpm₁₀₀ represents the counts bound inwells without inhibitor and cpm_(test) represents the counts bound inwells containing known amounts of FB or its analogs. Binding curves areconstructed for FB and its analogs by plotting % inhibition versusinhibitor concentration; th quantities of each analog required for 50%inhibition of binding are determined graphically. Binding constants ofthe reduced binding analogs are calculated as follows: ##EQU2## whereK(FB_(x)) is the binding constant for the analog FB_(x), K(FB) is thebinding constant for the native fragment B molecule (assumed to be 5×10⁷M⁻¹ Langone, 1982), and [FB_(x) ] and [FB] are the molar concentrationsof FB_(x) and FB required for 50% inhibition of binding.

Selective Binding of Immune Complexes using Monomeric Protein A Analogs

FB₅₈ (native FB) or a reduced binding analog is diluted in 0.1Mcarbonate buffer, pH 9.0, and adhered to the wells of polystyrenemicrotiter plates for 2 hr at 37° C. Varying the analog concentrationbetween 1.5 and 100 μg/ml has little or no effect on the subsequentbinding of ¹²⁵ I-labeled soluble IgG or heat aggregated IgG. Theremaining protein binding sites are blocked by incubation with 1% skimmilk for 1 hr. After removal of the blocking solution, soluble IgGdiluted in 1% skim milk is added for 2 hr at room temperature tosaturate immunoglobulin binding sites. The excess is removed by washingthe wells with BSB; varying concentrations of ¹²⁵ I-labeled soluble IgGor aggregated IgG (adjusted to the same specific activity) are added tothe wells and incubation is continued overnight. Comparison, for the twolabels, of absolute counts bound in wells coated with FB₅₈ or an analogis used to assess selective binding. A representative experimentcomparing the ability of FB₅₈, FB₂₉ and FB₄₀ (See FIG. 1) to bind ¹²⁵I-IgG and ¹²⁵ I-aggregated IgG is shown in FIG. 3. In wells coated withFB₅₈, similar and significant quantities of both ligands are bound atligand concentrations between 5 and 40 μg/ml and a saturationconcentration of soluble IgG of 1 mg/ml. Attempts to abrogate thebinding of ¹²⁵ I-IgG to FB₅₈ by increasing the saturation concentrationof soluble IgG to 5 mg/ml were unsuccessful (due to working around theequilibrium concentration of soluble IgG), although the absolute numberof counts bound was decreased. In wells containing FB₂₉ and FB₄₀, theabsolute number of counts of both ligands is decreased compared to FB₅₈-containing wells, but significantly greater quantities of ¹²⁵I-aggregated IgG is bound compared to ¹²⁵ I-soluble IgG. In thisconfiguration, more counts are bound in wells containing FB₄₀ than FB₂₉.Increasing the saturation concentration of soluble IgG from 1 to 5 mg/mlenhanced selective binding in wells coated with the analogs.

SELECTIVE REMOVAL OF IMMUNE COMPLEXES USING IMMOBILIZED MONOMERICPROTEIN A ANALOGS Immobilization

Sepharose CL-4B (Pharmacia) is activated as follows. The gel is washedsequentially with water, dioxane/water mixtures, and anhydrous dioxaneprior to the addition of anhydrous recrystallized4-dimethylam:inopyridine (DMAP). A solution of tosyl chloride inanhydrous dioxane is then added and the mixture is shaken at roomtemperature for 15 min. The mixure is filtered with anhydrous dioxane toremove any unreacted DMAP and tosyl chloride. A 0.5 M solution ofdiaminodipropylamine (DADPA) in anhyrdrous dioxane is then added and themixture is shaken overnight under nitrogen at 4° C. The gel is filteredand sequentially washed with anhydrous dioxane, dioxane/1 mM HClmixtures, and finally water. After additional washing with 0.1M sodiumphosphate buffer, pH 6.7, containing 10 mM EDTA, a freshly made solutionof the heterobifunctional cross-linker, m-maleimidobenzoylsulfosuccinimide (sulfo-MBS) is added and the gel is mixed for 2 hr atroom temperature. The gel is washed again with 0.1M sodium phosphate, 10mM EDTA, pH 6.7 and then with 0.1M sodium acetate, 10 mM EDTA, pH 5.0.The activated gel with the attached sulfo-MBS is separated intoaliquots, and mixed with solutions containing various concentrations ofa Protein A analog. The mixtures are agitated at room temperature for 90min, and washed with sodium acetate-EDTA buffer, pH 5.0. Unreactedsulfo-MBS is blocked for 90 min by the addition of 0.1M2-mercaptoethanol in the same buffer. After blocking, the gel is washedwith sodium acetate-EDTA buffer, pH 5.0, and then with 10mM sodiumphosphate, 150 mM NaCl, 2 mM EDTA, pH 7.3. The gel is stored at 4° C. inthe sodium phosphate buffer with 0.02% sodium azide until use.

Immune Complex Assay

Complex is quantitated using the enzyme-linked immunoassay kit marketedby Cytotech according to the manufacturer's instructions. Threestandards of heat-aggregated IgG, are included in the kit to allow theconstruction of a standard curve. The values of unknown samples aredetermined from the standard curve; sera containing less than 4 μgequivalents (Eq)/ml are considered normal, while those with higherlevels are considered elevated.

Soluble Human IgG RIA

Soluble IgG levels are quantitated by RIA. Briefly, specificallypurified goat anti-human-IgG, diluted to a concentration of 5 μg/ml, isabsorbed to the wells of PolYvinyl chloride microtiter plates. Afterblocking non-specific protein binding sites with 1% lowfat dry milk,aliquots of IgG standards or dilutions of unknown samples are added tothe well and the plates are incubated at room temperature for 4 hr.Following extensive washing to remove unbound material, a constantamount of ¹²⁵ I-labeled goat anti-IgG is added to each well. At the endof an 18-24 hour incubation at room temperature, the plates are washed,dried, cut and counted. A standard curve is constructed using samples ofknown soluble IgG concentrations between 7.5 and 640 ng/ml. Unknownsamples are assayed in triplicate wells at four different dilutions. Theconcentration of IgG in an unknown is calculated from the standardcurve; those dilutions falling on the linear portion of the curve arecorrected for dilution and averaged to obtain the reported IgGconcentration. This assay detects IgG in complex, in addition to sol-Ab,although complex is detected less efficiently on a μg/ml basis.

Selective Removal of Circulating Immune Complex Using Sepharose-CoupledProtein A Analogs

Aliquots of each Sepharose-FB analog preparation are placed in Eppendorfcentrifuge tubes. The gel is washed twice and the supernatantsdiscarded. One hundred μl samples of chemically aggregated IgG dilutedin normal human serum (CAG/NHS) or normal human serum similarly dilutedwith buffer (NHS) are added to the gel, mixed and incubated for varyingamounts of time at 37° C. Similar results are obtained when the time ofadsorption is varied between 5 and 120 min and the temperature ismaintained at 37° or 25° C. The supernatants are removed to separatetubes, and the gel samples are washed by the addition of 100 μl ofbuffer. The wash is pooled with the initial supernatant. Control samplesare absorbed on Sepharose to which no Protein A analog has been coupled.Subsequently, each sample is diluted appropriately and complex andsol-Ab levels are determined using the Cytotech EIA kit and IgG RIA,respectively.

Table I set forth below presents the results of an experiment in whichthe effect of concentration of immobilized analog on selective removalof immune complexes was determined.

                  TABLE I                                                         ______________________________________                                                            CIC Level,   HuIgG Level,                                 Sepharose Sample    μg/ml     mg/ml                                        ______________________________________                                        None      CAG/NHS   21.0           4.03                                       Control   CAG/NHS   18.3     [13].sup.1                                                                          3.70  [8]                                  FB.sub.58, 4 mg/ml                                                                      CAG/NHS   8.0      (56).sup.2                                                                          0.90  (76)                                 FB.sub.58, 3 mg/ml                                                                      CAG/NHS   7.0      (62)  0.99  (73)                                 FB.sub.58, 2 mg/ml                                                                      CAG/NHS   9.1      (50)  1.49  (60)                                 FB.sub.58, 1 mg/ml                                                                      CAG/NHS   10.2     (43)  2.11  (43)                                 FB.sub.29, 4 mg/ml                                                                      CAG/NHS   7.0      (62)  3.55  (4)                                  FB.sub.29, 3 mg/ml                                                                      CAG/NHS   8.0      (56)  3.72  (-1)                                 FB.sub.29, 2 mg/ml                                                                      CAG/NHS   10.3     (44)  3.04  (18)                                 FB.sub.29, 1 mg/ml                                                                      CAG/NHS   12.7     (31)  3.73  (-8)                                 None      NHS       2.2            3.82                                       Control   NHS       1.6            3.10  [19]                                 FB.sub.58, 4 mg/ml                                                                      NHS       0.2            0.92  (70)                                 FB.sub.58, 3 mg/ml                                                                      NHS       ND.sup.3       1.04  (66)                                 FB.sub.58, 2 mg/ml                                                                      NHS       ND             1.07  (65)                                 FB.sub.58, 1 mg/ml                                                                      NHS       ND             1.97  (36)                                 FB.sub.29, 4 mg/ml                                                                      NHS       0.8      3.39        (-9)                                 FB.sub.29, 3 mg/ml                                                                      NHS       ND             3.26  (-5)                                 FB.sub.29, 2 mg/ml                                                                      NHS       ND             3.46  (-13)                                FB.sub.29, 1 mg/ml                                                                      NHS       ND             2.78  (10)                                 ______________________________________                                         .sup.1 Number in brackets represents the % reduction compared to an           unadsorbed sample similarly handled.                                          .sup.2 Number in parentheses represents the % reduction compared to the       sample absorbed on control Sepharose.                                         .sup.3 Not determined.                                                   

Absorption of CAG/NHS with each preparation of Sepharose-FB₅₈ or FB₂₉decreased complex concentration measurable in the cotnrol sample from18.3 μg Eq/ml to between 7 and about 13 μg Eq/ml. Analog concentrationsof 3 and 4 mg/ml of gel are capable of removing larger quantities ofcomplex compared to lower analog concentrations. After adsorption of NHSon gel derivitized with FB₅₈, human sol-Ab levels are decreased to36-70% of control values. In contrast, adsorption of samples of NHS onFB₂₉ derivitized gel resulted in little or no decrease in levels ofsol-Ab (-13% to 10% reduction).

A subsequent experiment was performed to determine whether complexlevels could be decreased in a dose dependent manner by adsorption withinceasing quantities of immobilized Protein A analogs. Volumes ofconjugated Sepharose, between 25 and 200 μl of packed gel, weredistributed into tubes. The analog to gel concentration of both FB₅₈ andFB₂₉ in this experiment was 4 mg/ml gel. One hundred microliter samplesof CAG/NHS were added to each tube, mixed and incubated at roomtemperature for 10 min. The supernatants were removed, and the gelsamples washed and processed as described above. The results of thisexperiment are presented in Table II which is set forth below, whereinCIC indicates circulating immune complex and HuIgG indicates humanimmunoglobulin.

                  TABLE II                                                        ______________________________________                                                Control     FB.sub.58 -  FB.sub.29 -                                  Volume  Sepharose   Sepharose.sup.1                                                                            Sepharose.sup.1                              Sepharose                                                                             CIC     HuIgG   CIC    HuIgG CIC   HuIgG                              ______________________________________                                         25 μl                                                                              29.68.sup.2                                                                           4.25.sup.3                                                                           21.52  2.65  20.40 4.68                                                        (27).sup.4                                                                          (38)  (31)  (-10)                               50 μl                                                                             30.92   4.43    19.60  1.75  12.40 3.73                                                       (37)   (60)  (60)  (16)                               100 μl                                                                             28.68   3.57    13.20  0.77  10.12 4.19                                                       (54)   (78)  (65)  (-17)                              150 μl                                                                             23.68   3.64     5.60  0.46   5.92 3.56                                                       (76)   (87)  (75)  (2)                                200 μl                                                                             26.40   2.99     2.72  0.34   4.92 3.01                                                       (90)   (89)  (81)  (-1)                               ______________________________________                                         .sup.1 The concentration of analog to gel used in these experiments was 4     mg/ml.                                                                        .sup.2 CIC levels are reported in μg Eq/ml.                                .sup.3 HuIgG levels are reported in mg/ml.                                    .sup.4 The values in parentheses are the percent reduction in levels          compared to the same volume of control Sepharose.                        

Absorpiton of CAG/NHS on increasing quantities of FB₅₈ - or FB₂₉-Sepharose resulted in a dose dependent reduction in CIC levels, form 27to 90% of control values. Sol-Ab levels were similarly decreased afteradsorption on Sepharose-FB₅₈ ; between 38 and 89%. In contrast, thesoluble IgG levels of samples absorbed on Sepharose-FB₂₉ showed littleor no reduction (between -17 and 16% of control values).

The invention may be embodied in other specific forms, and otherembodiments are within the following claims.

What is claimed is:
 1. A DNA molecule which encodes a polypeptidecomprising plural, spaced apart binding domains of protein A or proteinG,individual said domains binding with a site on human Fc with a bindingconstant less than 10⁷ M⁻¹, and the spacing of said domains being suchthat a plurality of said domains together bind to a correspondingplurality of sites on plural human immunoglobulins aggregated in acomplex by multiple point attachment with a net binding constant greaterthan 1×10⁷ M⁻¹.
 2. A DNA molecule of claim 1 wherein at least one ofsaid domains comprises a polypeptide homologous to an Fc bnding portionof Staphylococcal Protein A.
 3. A DNA molecule of claim 1 encoding acysteine residue adjacent the amino terminus of said polypeptide.